Aromatic oligoesters as novel helix mimetic scaffolds

Graphical abstract


Introduction
The interactions between protein binding partners critically mediate almost all biological processes, with the size of the human protein-protein interaction (PPI) interactome thought to be ~ 650,000 pair-wise interactions. 1 As such, considerable effort has been directed towards PPI modulation and inhibition over recent decades with α-helix mediated interactions garnering the most attention. 2 Multiple strategies for targeting helix-mediated PPIs have emerged including small molecule screening, 3 peptidic 4,5 and peptidomimetic (or proteomimetic) approaches. [6][7][8] The latter involves the development of molecules able to replicate the spacial projection of the binding amino acid side chains on an α-helix from a central scaffold. Many such helix-mimetic scaffolds have been developed since the initial terphenyls [9][10][11][12] to now encompass oxopiperazines, 13 oligoureas 14 and triazine-piperazine-triazine scaffolds, 15 among others. [16][17][18] However, it is aromatic oligoamides 19,8 which have dominated this space owing to their synthetic tractability and amenability to automated solid-phase synthesis [20][21][22][23] with examples showing promising competitive inhibition of disease relevant PPIs. [24][25][26][27][28][29][30][31][32] Despite their dominance, aromatic oligoamide scaffolds are not without drawbacks; the poor nucleophilicity of anilines in combination with weakly electrophilic benzoic acids necessitates harsh coupling conditions 23 and substituted anilines do not always adopt a mimetic conformation without internal hydrogen bonding interactions which reduce aqueous solubility. 33,34 We reasoned that perhaps some of these issues could be circumvented by replacing the central amide bond with an ester linkage (Fig. 1). This would enable coupling between monomers with more reactive phenol nucleophiles (vs aniline) and would take advantage of the conformational preference of esters for an extended, mimetic s-cis orientation. Herein, we describe the design, synthesis, and analysis of a novel 3-O-alkylated aromatic oligoester helix mimetic scaffold capable of effective mimicry of a canonical α-helix and complete stability to degradation at biologically relevant pH.

Monomer synthesis
Our oligoester scaffold was designed to minimise synthetic steps, avoid complex chemistry and enable efficient generation of a range of different 'side chain' functionalities. Taking inspiration from established routes to oligoamide scaffolds, 21 we chose to start from methyl 3,4-dihydroxybenzoate and install a protecting group para to the methyl ester and subsequently introduce side-chains at the meta-hydroxy to afford a library of protected monomer building blocks (Scheme 1). We chose to incorporate a para-allyl protecting group based on its low steric hindrance, minimal electronic effects and, primarily, acid and base stability allowing for orthogonal deprotection of the methyl ester and any acidlabile side chain protecting groups. Importantly, this leaves open the possibility of expansion of our methodology in future to automated solid-phase synthesis allowing for future global side chain deprotection and resin cleavage. To install the allyl protecting group, two sets of conditions were trialled: substitution with allyl bromide and Mitsunobu conditions with the former offering superior yields and more straightforward purification. Despite the electron-withdrawing effect of the ester group, at larger scales di-allyl protection was observed. Therefore, conditions were optimised through adjustment of the quantity of base to minimise this but the undesired bis(allyloxy) by-product was still formed necessitating purification. Nevertheless, the reaction could still be performed on a large (50 mmol) scale affording gram quantities of a common starting material for all monomer building blocks. This provides an advantage over oligoamides in which the Fmoc protecting group at the equivalent position is installed after the side chain is introduced. 21 With the singly protected monomer obtained, NOESY NMR was used to confirm isolation of the para-allyl protected compound 1 (Fig. S1).
Side chain functionality could then be introduced at the meta position using a range of bromides or alcohols employing substitution or Mitsunobu chemistry, respectively. Fourteen of the twenty natural amino acid side chains were initially considered: glycine and alanine were discounted as they do not form typically form meaningful interactions, glutamic acid and glutamine were considered represented with aspartic acid and asparagine mimics respectively, cysteine derivatives could prove problematic given their disulphide bond forming ability and proline is not commonly found within helical protein regions, acting instead as a helix breaker. Unfortunately, problems were encountered when attempting to introduce mimics of both tyrosine and arginine. Both tertbutyl (4-hydroxylmethyl)phenyl) carbonate and di-Boc protected 1-(3-hydroxypropyl)guanidine were unreactive under Mitsunobu conditions and generation of the equivalent bromide derivatives also proved unsuccessful. However, using our optimised conditions, it was possible to generate a library of monomers mimicking twelve of the proteogenic amino acids ( Table 1).
The final step in the synthesis of the aromatic oligoester monomers was ester hydrolysis which was readily achieved for most cases using either strong (NaOH) or weak (LiOH) base selected based on side-chain sensitivity. However, in the case of aspartic acid mimic 12 this resulted in side chain deprotection and for tryptophan mimic 22 side chain cleavage, likely via Boc deprotection in a similar manner to that previously reported for oligoamide helix mimetics. 22 For threonine derivative 23 no reaction occurred.

Dimer synthesis
With a library of monomer building blocks in hand, we sought to develop conditions for the synthesis of a representative dimer which could be readily applied for the generation of longer oligomers both in solution and on solid phase. Initially a dimer was made based on monomers furnished with isopropyl side chains. First, the allyl group on monomer 2 was removed using Pd(PPh 3 ) 4 and K 2 CO 3 affording phenol 24 which could then be coupled to carboxylate 3 using ester coupling conditions (EDC, HOBt and DIPEA) to afford the target dimer (25) in excellent yields (Scheme 2). To facilitate conformational analysis, a second dimer based on methyl side chains was also generated starting from commercially available methyl 4-hydroxy-3-methoxybenzoate which was subjected to para-allyl protection and subsequent methyl ester deprotection before being coupled using the same conditions established for dimer 25 to afford methyl functionalised dimer 28. Interestingly, when we attempted to remove the allyl protecting group from this dimer (28) using the conditions established for monomer 24, a transesterification with the methanol solvent occurred. We therefore switched to using the same palladium catalyst (Pd(PPh 3 ) 4 ) but in combination with NaBH 4 as a reducing agent to synthesise dimer 29.

Conformational analysis
To study the conformational preferences of the oligoester scaffold dimers 25 and 28 were subjected to a combination of solution and solidstate investigations since both contain all the structural features common to any extended oligomers: (i) a central ester bond and (ii) the potential for syn/anti conformers with regards to the O-alkyl side chains. Clearly, an extended ester and side chain syn conformation is optimal for effective mimicry of the binding groups along a single face of an α-helix.
Single crystals suitable for X-ray diffraction were obtained for dimer 28 from slow evaporation of ethyl acetate (Figure 2a). A single conformer was observed in the solid state with, pleasingly, the s-cisisomer about the central ester bond. This is to be expected due to the stabilising influence of an anomeric-type effect (nO sp2 → σ* C -O ), reduction in steric strain and the smaller dipole moment of the s-cisisomer. Single crystals of 25 could not be obtained so a variable temperature (VT) NMR experiment was carried out to determine whether multiple conformations of the scaffold could exist in solution. Over a temperature range of 218-328 K in CDCl 3 only a single conformer was observed for this dimer ( Figure S2). Based on the solid-state structure of 28 and the known conformational preferences of esters we concluded that our oligoester scaffold exists in a single stable extended conformation likely capable of effective helix mimicry. The solid-state structure also showed the side chains of 28 in an anti-conformation which would require rotation about the central aryl-C(O) bond to form a 'mimetic' arrangement of binding groups. We therefore calculated the barrier to rotation about this bond and confirmed that rapid bond rotation at room temperature is feasible (6.22 kcal/mol). This is also confirmed by our observation of a single conformer by VT NMR suggesting that this bond rotation is fast on the NMR timescale at all temperatures studied.
To confirm the ability of our scaffold to present side chains at similar orientations to those on a canonical α-helix we overlayed a computationally generated trimer structure with the i, i + 4 and i + 7 residues of the published crystal structure of the Bak peptide helix 35 and obtained an RMSD of 0.83 Å demonstrating good agreement (Figure 2b). These values are also similar to those obtained for oligoamide helix mimetics (~0.49 Å) 27,36 suggesting that similar PPI inhibition behaviour would be possible for our oligoester analogues.

Aqueous stability
A vital property any biologically active molecule must possess is aqueous stability. Human serum pH is tightly regulated around 7.4, with intracellular pH controlled between pH 6.0-7.2, and gastric pH between 1.4-3.5. 37 Thus, for oligoesters to serve as effective PPI inhibitors they must demonstrate sufficient stability at such pH ranges, to be capable of reaching the target without suffering from degradation that ultimately eliminates any activity. Given the prevalence of ester linkages in prodrug structures 38 and the facile hydrolysis of esters in aqueous media, the stability of our scaffold was a concern. We therefore investigated the stability of dimers 25 and 29 over extended periods of time in aqueous solution from pH2.5-12.5. To monitor dimer stability over time, 10 mM pH buffer solutions of: 2.5(acetate), 5.0(acetate), 7.4 (phosphate-buffered saline), 10.0 (bicarbonate), and 12.5 (bicarbonate) were selected, to obtain a pH range similar to those present within the human body. Since both dimers are insoluble in water, they were firstly dissolved in acetonitrile, before addition to the buffered solutions to form a 5 mg/mL dimer solution (50% MeCN: buffer). Degradation was monitored via TLC over time, and the final solution composition was determined by LCMS after 7 days (Fig 3 and Figure S3).
Promisingly, we detected no significant degradation of either dimer in the pH range 2.5 -10.0, even after 7 days, suggesting the ester bond has strong pH stability with dimer % remaining consistently over 90% (i. e., less than 10% ester hydrolysis) when determined from the corresponding HPLC UV traces. Strong stability at pH 7.4 is particularly important, indicating the ester bond would be capable of travelling within human serum without suffering from degradation. Additionally, pH 2.5 stability means the ester bond should be capable of surviving the acidic conditions of the stomach without succumbing to hydrolysis. Such results should also be applicable to trimers or longer oligoesters since the core nature of the ester bond would remain un-changed even in extended chains. However, dimer degradation, via ester bond cleavage, does occur at pH 12.5 after 7 days, with dimer % overall decreasing to 6% for 25 and 49% for 29. Some drift to lower pH from 12.5 was observed for 29, suspected to be the result of deprotonation of the phenol, which accounts for the higher stability observed for this dimer. Further analysis of dimer 25 ( Figure S3) confirmed that the one original UV-active peak corresponding to starting dimer, degraded to two monomer peaks with m/z appropriate for the phenol and carboxylic acid by-products resulting from ester hydrolysis. Nonetheless, this hydrolysis occurs only after 5 days at pH 12.5, an acceptable duration of ester bond stability particularly at this extreme basicity, beyond the range any inhibitor would encounter in a biological environment.

Conclusions
We have described the design, synthesis, and analysis of a novel aromatic oligoester scaffold capable of mimicry of the side chains i, i + 3/4 and i + 7 of a canonical α-helix. We have developed a facile synthetic route to access a range of monomer building blocks covering a variety of side chain functionalisation similar to the diversity of those found in natural amino acids and, importantly, our methodology is readily expandable to encompass the vast chemical space not sampled in native proteins. Conformational analysis demonstrated a favourable extended, mimetic conformation for the ester scaffold and good agreement between the spacial projection of side chains on a model α-helix and those projected from our scaffold backbone. The ester linkage was found to be surprisingly stable over a range of pH values (2.5-10) making the application of this class of helix mimetic in a biologically relevant environment highly feasible. With further development of higher-throughput automated solid-phase synthetic methods, oligoester scaffolds could potentially offer a viable alternative to current oligoamide analogues overcoming some of the issues with their synthetic accessibility and non-mimetic conformational preferences.

General considerations
All reagents were obtained from commercial sources unless otherwise stated. All solvents used were anhydrous. All water used was of distilled (dH 2 O) or Milli-Q quality. Solvents were removed using Büchi rotary evaporators. Reactions were conducted under inert (nitrogen) atmosphere. Column chromatography purification was performed using silica gel (40-63 μM, Geduran) with the solvent ratios specified. Reaction monitoring was done using Thin Layer Chromatography (TLC) on silica Merck 60 F254 aluminium plates, using Ultraviolet (UV) 254/365 nm for visualization. 1 H, 13 C, COSY, and NOESY spectra were all measured on a Bruker DRX400, with chemical shifts reported in parts per million (ppm) downfield from trimethylsilane (TMS) as the internal reference, coupling constants are reported in hertz (Hz). Variable temperature NMR was conducted between 218 K and 328 K on a Bruker DRX500. Multiplicity is reported as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), or combinations of these. Infrared spectra were obtained using a Perkin-Elmer Spectrum 100 FTIR spectrometer, reported in cm − 1 , and obtained in solid-state. High-resolution mass spectrometry (HRMS) of products were obtained with an in-house highresolution mass spectrometer (Waters LCT Premier Electrospray Time of Flight spectrometer), using an electron spray ionization (ESI) technique. Melting points were obtained using a Büchi 510 melting point machine, with a gradient of 1 • C per minute. X-ray crystallography was performed by the Imperial College X-ray Crystallography Service. Single crystal Xray data were collected using an Agilent Xcalibur 3 E diffractometer with Mo-K(alpha) radiation at 173 K.

Molecular modelling
Superimposition of the oligoester with the Bak peptide α-helix was conducted using MOE2022.02 software. The oligoester was energy minimized using the Amber10:EHT forcefield. The central ester bond was then selected and, using the bidirectional torsion profile, conformational analysis was conducted. This gave a standard sin(θ) curve for the dihedral angle ranging from − 180 • to 180 • across this bond.
The Bak peptide was imported into MOE using PDB ID 1BXL. The inbuilt 'QuickPrep' function was used to allow for energy minimisation and side-chain resolution. The oligoester was then superimposed, using the 'superpose molecules' function such that the oligoester oxygen atom of the OMe aligned with the α-carbon of the side chain in the Bak peptide. This was done at the i, i + 4, i + 7 positions of the α-helix. In addition, the carbon atom in the central phenyl ring of the oligoester 'backbone' was superimposed with the corresponding backbone carbonyl of the Bak peptide to ensure the correct directionality was obtained. Using the SVL function, a RMSD value of 0.83 Å was obtained.

Aqueous stability
To pre-prepared 100 mM pH buffer solutions (1.00 mL), including pH 2.5, 5.0, 7.4, 10.0 and 12.5 (Table 2), was added the dimer (10 mg/ mL in MeCN) at r.t, forming a final solution with dimer concentration 5 mg/mL. The mixture was stirred at r.t. for 7 days and monitored via TLC and LCMS (50% MeCN) to follow ester bond degradation.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability
All raw data has been uploaded onto the Imperial College Data Repository, the DOI is shared in the manuscript.